Stem cell capture and immobilization coatings for medical devices and implants

ABSTRACT

Constructs and methods for immobilizing stem and other precursor cells, as well as other bioactive materials of therapeutic value on the surfaces of medical devices, such as bone, cartilage, spinal and tooth implants, are described herein. The present invention has broad application in the incorporation of bioactive and therapeutic materials in or on a medical implant or other interventional device, having particular value in enabling the real-time, utilization by medical personnel of bioactive materials extracted from the patient and subsequently reintroduced and immobilized in an implant device.

PRIORITY

The instant application is a divisional of U.S. patent application Ser.No. 12/537,409 filed Aug. 7, 2009, which, in turn, claims the benefit ofU.S. Provisional Application Ser. Nos. 61/086,912 filed Aug. 7, 2008,and 61/1,153,076 filed Feb. 27, 2009, the entire contents of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the immobilization of bioactivematerials, such as stem cells, other biological cells, bioactivemolecules, particularly growth factors, and other materials oftherapeutic value, on internal and/or external surfaces of medicaldevices, particularly porous implants where bone or tissue ingrowth isdesired. In one particular embodiment, the present invention utilizes ahigh-density nanofilm of amphiphilic molecules to selectively capture,concentrate and immobilize the bioactive material, particularly cellularmaterial, of interest. Such high-density nanofilms, embedded withselective recognition molecules or targeting moieties, may be formed onthe surface of a medical device.

The novel constructs and methods of the present invention findparticular utility in an operating room environment, enabling medicalpersonnel to coat a prefabricated medical device, such as a boneimplant, just prior to use and particularly to utilize bioactivematerials extracted from the patient (e.g., autologous and/or endogenoustransplant materials), thereby reducing concerns about shipment andstorage of bioactive materials as well as adverse immunologicalreactions caused by genetic incompatibilities or transmission ofinfective agents.

BACKGROUND OF THE INVENTION

Porous medical implant devices, particularly of metallic, ceramic orpolymeric construction, but also those of biological origin, have provenof great value as scaffolds for tissue growth in medical applications.Such constructs find particular utility as scaffolds for bone growth,where the porous structure allows the prosthetic device to bind withadjacent bone as cartilage and bone grow into the pores of the device.

Many techniques have been proposed to promote the desired tissueingrowth, including the incorporation of molecules that stimulate tissuegrowth, such as growth factor proteins, into the pores of the prostheticdevice prior to implantation of the device in the patient. Thesetechniques typically involve surface coating, adsorption onto a metallicsurface, conjugation to a polymer surface or void-filling withbiodegradable materials. Plastics such as PLA and PEG find particularuse in these void-filling applications, although the degradationproducts of these materials in any significant quantity can impairbiological function. Fibrin, collagen and bone-based cements have alsobeen used in these void-filling applications. Other techniques forimmobilizing bioactive materials in the porous structure utilizingvarious types of coatings have also been proposed.

The many examples in the art where therapeutic and/or bioactivematerials are incorporated into medical devices are often focused on theuse of a limited number of predetermined types of bioactive molecules,such as specific growth factors, which have been produced in a sterileproduction environment, often by recombinant techniques. Such coatingsystems are often better suited to a manufacturing environment than asurgical operating room. Additionally, growth factors and othertherapeutic materials are found to have complex interactions with eachother, all of which are not clearly understood even by those skilled inthe art. While selected growth factors introduced from syntheticproduction have proven to have benefit, they are costly to produce andcan provide adverse reactions in the patient. In addition, the selectedmix of components may not have the range of therapeutic activities thatmay be present in endogenous tissues and fluids, such as bone marrow andadipose tissue. Because of this, in certain procedures, surgeons willoften extract tissues or fluids from a patient, put it through aseparation process, such as centrifugation, select a fraction which isknown to be rich in desired materials, such as growth factors, stemcells or progenitor cells, and then re-inject that material into thepatient at a point of injury or surgical intervention to promotehealing. The present invention is directed towards the capture andutilization of bioactive molecules and biological cells as might bepresent in a patient's own tissues and fluids, though the novelconstructs of the present invention are also compatible with the use ofsynthetically produced bioactive molecules and biological cellsharvested from cell cultures.

Bone marrow for clinical use is typically obtained as an aspirateextracted from a target patient's bone using a syringe-type device.Often the iliac crest, pelvis or pelvic bone is used as a source due toits large size and proximity to the surface of the body. In someapplications, the bone marrow is used without modification, but in manycases some form of separation technology, such as centrifugation, isused to concentrate the desired fraction of the bone marrow. Stem cellsand bioactive molecules, including cytokines such as growth factors, areoften the target of this separation process, though separation throughcentrifugation tends to select a fraction that also contains a highlevel of white blood cells and a broad spectrum of molecular components.Cells and molecules of interest can also typically be obtained fromadipose, also fat, tissue. Any tissue of the body has potential, muscleand nerve tissue and tissues associated with the reproductive processare also of particular interest. Material extracted from the patient orintended recipient (i.e., autologous transplant material) has severaladvantages over other sources, including inherent biocompatibility,potential for lower cost, providing a broader spectrum of usefulcompounds that might have synergistic effects and potentially reducedregulatory issues or faster regulatory approval. When bone marrowderivatives are used in surgery, they are typically reintroduced intothe body by injection by syringe into an area of desired activity orinto an implant device or scaffold material which is then implanted inthe body.

Many of the current techniques of immobilizing bioactive materials onmedical devices, such as prosthetic bone implants, are not well-suitedto allowing surgical teams to exercise an option to use endogenousbioactive materials. A construct which allows the surgical team toremove bone marrow from the patient and concentrate and immobilizeselected bioactive components of such in the device provides advantagesin genetic compatibility of the material as well as potentially reducedcost. The key challenges in such a system are the incorporation of amostly liquid material into a highly porous material and the retentionthere while the device is being handled and implanted in the patient.

Thus, the present invention addresses a need in the art, providing forthe capture and delivery of bioactive molecules and particularly thereal-time utilization of extracted tissues and fluid, whether from theintended recipient (i.e., autologous transplant materials) or a selecteddonor organism (i.e., allogenic, homologous or heterologous transplantmaterials), as well as materials that are synthetically produced orproduced from cell cultures (recombinant transplant materials). Inparticular, embodiments of the medical device constructs, kits andpackaging systems of the present invention have unique and valuableadvantages over current art and enable new medical techniques, withparticular importance in surgical procedures.

SUMMARY OF THE INVENTION

As noted above, there are no readily available systems in the art for onsite treatment of medical devices, such as prosthetic implants, to allowbioactive materials such as stem cells to be immobilized andconcentrated on their surfaces, despite the acknowledged benefitsthereof. Herein, it was discovered that disposing an amphiphilic film onthe surface of a medical device, with a non-polar liquid/film acting asa “binder” therebetween, facilitates the capture, concentration andimmobilization a targeted therapeutic cell or molecule in an efficient,expeditious and economical manner. In the context of the presentinvention, a plurality of amphiphilic molecules spontaneously align atthe interface of a relatively non-polar surface or substrate and arelatively polar surrounding environment and assemble into a molecularlythin, extremely dense, and well-oriented film coating. By affording thehydrophilic head of at least some of the amphiphilic molecules with atargeting moiety having a binding affinity for one or more targetbioactive material of interest, the present invention enables the rapidextraction and immobilization of such bioactive material upon exposurethereto.

Accordingly it is an object of the present invention to provide abiocompatible device comprising a solid surface having a film ofnon-polar liquid disposed thereon, the non-polar liquid film having aplurality of amphiphilic molecules disposed as a monolayer thereon,wherein at least one of the amphiphilic molecules includes orincorporates at least one targeting moiety having binding affinity forbioactive material of interest, for example a target molecule or asurface moiety of a target cell.

The present invention contemplates the use of different amphiphilicmolecules and/or targeting moieties, having divergent bindingaffinities, in a single device, so as to enable the capture of aplurality of different bioactive materials, particularly materialshaving synergistic functionality (e.g., stem cells and growth factors).By the same token, the present invention also contemplates the inclusionof different targeting moieties that target different structures of thesame bioactive material (e.g., different epitopes, surface peptides,adhesion molecules, etc.).

In a preferred embodiment, the targeting moiety is a nucleic acidaptamer, antibody, or a product of a phage-display technique. In aparticularly preferred embodiment, the amphiphilic molecule is anaptamer conjugated to a hydrocarbon chain of the form (CH2)n where n isgreater than eight. Alternatively, the amphiphilic molecule is aconjugation of biotin, avidin and either an aptamer or antibody.

As discussed in detail below, although the present invention findsparticular utility in the context of prosthetic implants, it is readilyunderstood that the concepts may be extended to other medical devicesand biocompatible structures. In a similar fashion, although the presentinvention finds particular utility in the context of biological cells,such as stem, precursor and differentiated cells, as well as a widerange of graft and transplant materials, including autologous,homologous and heterologous transplant materials such as bone marrow andconnective tissues, the concepts of the present invention are notlimited thereto and may be readily applied to the capture of othertarget cells and molecules, for example pathogen cells and bioactivepeptides such as growth factors.

It is a further object of the present invention to provide sterile kitsand packaging systems, for example as a kit adapted for the constructionof a bioactive material-immobilizing coating including:

-   -   a. a sterile solution of a polar liquid and amphiphilic        molecules, wherein the amphiphilic molecules include at least        one targeting moiety having binding affinity for a bioactive        material of interest, such as a target molecule or a surface        moiety of a target cell; and    -   b. a sterile, relatively non-polar liquid.

In addition or alternatively, the device or kit components of thepresent invention may be bundled in a sterile package that facilitatescontact between the biocompatible device and the bioactive material ofinterest. For example, the package may be adapted to permit the passageof a biological fluid through an interior surface of the device.

It is yet a further object of the present invention to provide methodsof making and using the components of the present invention. To thatend, the present invention provides for the construction of a medicaldevice having a bioactive material-immobilizing coating disposed thereonby:

-   -   a. providing a biocompatible medical device;    -   b. contacting the medical device with a non-polar solution to        yield a medical device having a non-polar film coating disposed        thereon;    -   c. contacting the coated medical device of step b with an        amphiphilic molecule-containing polar solution, either in        conjunction with step b or after step b, to yield a medical        device having a non-polar film coating disposed thereon, the        film coating having a monolayer of amphiphilic molecules        disposed thereon;    -   d. optionally contacting the coated medical device of step c        with a polar rinse solution; and    -   e. exposing the coated medical device to a second polar solution        containing one or more bioactive materials of interest, such as        target molecules or cells to which the targeting moieties of the        amphiphilic molecules have a binding affinity, to yield a        medical device having a non-polar film coating disposed thereon,        the film coating including said amphiphilic molecules disposed        as a monolayer thereon, the amphiphilic molecules being bound to        said target molecules or cells.

In a preferred embodiment, the second polar solution is or is derivedfrom a subject-extracted tissue sample, for example a graft ortransplant material including autologous, homologous and heterologoustransplant materials such as bone marrow and connective tissues.

As noted above, although the present invention finds particular utilityin the context of stem-cell coated prosthetic implants, it is readilyunderstood that the concepts may be extended to other medical devicesand biocompatible structures and the capture of other target cells andmolecules.

In addition to serving as a substrate for bioactive material capture,the amphiphilic films of the present invention also find particularutility as coatings for porous implants. The liquid-based systems of thepresent invention have the unique ability to create films on otherwiseinaccessible surfaces. Additionally, where films of the presentinvention can form non-polar-liquid-filled micelles, micelles of thistype tend to break down or coalesce when introduced into a porousconstruct. With a low density of micelles, the films just move to thewalls and coat them. With a higher density of micelles, the micellescoalesce with each other or partly with the wall, creating a moreviscous fluid that effectively “clogs” the pores of the device. Ifbioactive material is bound to the micelles as they are introduced intothe pores, the coalescence of the micelles and clogging of the poreswill result in a reduction in undesirable circulation or flow ofbioactive material out of the pores of the device.

In a broader sense, it is also an object of the present invention toprovide a medical implant device composed of a solid porous materialwherein the above-described or other biocompatible, viscous materialsare utilized in certain pores of a porous device and not in other pores,in a manner that, when in vivo, permits early ingrowth of tissue intocertain porous surfaces of an implant device and not others.Accordingly, a device of this design will have a first external surfaceand a second external surface, wherein the first external surface iscoated with (also the pores of that surface have embedded within them) abiodegradable viscous or solid material that impedes flow of materialacross said first external surface making the second external surfacethe more conducive avenue for tissue ingrowth. In one preferredembodiment, a highly viscous material is embedded in a perimeter zone ofthe device, the first external surface, to act as a hydraulic barrierand to constrain a less viscous material, preferably a bioactivematerial, disposed in the center zone and in the second external surfaceof the device.

It is also an aspect of the present invention that viscous materials ofvalue can also comprise emulsions and foams. An emulsion or foam thatsupersaturated with oxygen has the potential to expedite cellproliferation and subsequent healing as the oxygen gradually diffusesfrom the emulsion or foam into the surrounding tissue. Introduction intoa porous prosthetic implant of an emulsion or foam where the oxygenconcentration is greater than 20% of the gas present in the emulsion orfoam is an aspect of the present invention.

It will be understood by those skilled in the art that one or moreaspects of this invention can meet certain objectives, while one or moreother aspects can meet certain other objectives. Each objective may notapply equally, in all its respects, to every aspect of this invention.As such, the preceding aspects can be viewed in the alternative withrespect to any one aspect of this invention. These and other aspects andfeatures of the invention will become more fully apparent when thefollowing detailed description is read in conjunction with theaccompanying figures and examples. However, it is to be understood thatboth the foregoing summary of the invention and the following detaileddescription are of a preferred embodiment and not restrictive of theinvention or other alternate embodiments of the invention. Inparticular, while the invention is described herein with reference to anumber of specific embodiments, it will be appreciated that thedescription is illustrative of the invention and is not constructed aslimiting of the invention. Various modifications and applications mayoccur to those who are skilled in the art, without departing from thespirit and the scope of the invention, as described by the appendedclaims. Likewise, other aspects, features, benefits and advantages ofthe present invention will be apparent from this summary and certainembodiments described below, and will be readily apparent to thoseskilled in the art. Such aspects, features, benefits and advantages willbe apparent from the above in conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments which follows:

FIGS. 1 a and 1 b are images of tantalum metal implants photographedusing a fluorescence microscope. In FIG. 1 a, the implant has beenexposed to amphiphilic molecules with fluorescently tagged aptamers inwhich no non-polar liquid is present. In FIG. 1 b, the metal implant isfirst exposed to oleic acid (an exemplary non-polar liquid) in a mannerthat forms a thin film on the surface before being exposed to thefluorescently tagged aptamers. As can be seen from these images,aptamers can be successfully immobilized on the surface of a metallicimplant using the present invention.

FIGS. 2 a, b and c are 4× microscopic images of human mesenchymal stemcells (MSC) on glass slides. All three slides were exposed to solutionswith the same concentration of stem cells. In FIG. 2 a, the slide hasreceived no additional treatment or coating. In FIG. 2 b, antibodieswith an affinity for MSC has been introduced into solution. In FIG. 2 c,the slide has been first treated in accordance with the presentinvention with a non-polar liquid film and an amphiphilic surface filmincorporating antibodies with an affinity for MSC. A four-fold increasein stem cell immobilization was realized when the slide was treated inaccordance with the present invention.

FIG. 3 depicts a suitable sequence for use of solutions of the presentinvention by a surgical team in an operating room environment to coat asurgical implant with a patients own stem cells, extracted at the timeof surgery.

FIG. 4 depicts a bone (1) with a Trabecular Metal™ prosthetic implant(2) inserted in a non-union. The circumferential band (3) of the implanthas been impregnated with a biodegradable wax that constrains liquidtransport to and from the interior through the ends of the implant (4),which abut the bone. The porous metal on the interior of the implant issaturated with bone marrow aspirate prior to the implant being insertedinto the point of non-union. The net result is that the bioactivematerials in the implant are constrained to interact with the adjacentbone at the points where ingrowth is being promoted.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to constructs and methods for immobilizingbioactive material, including stem and other precursor cells as well asother bioactive molecules of therapeutic value, on the surface(s) ofmedical devices, such as bone, cartilage, spinal and tooth implants. Theconstructs, devices, kits and methods of present invention describedherein have broad application to the incorporation and/or immobilizationof bioactive material in or on a medical implant or other interventionaldevice, having particular value in enabling the utilization by medicalpersonnel of bioactive materials extracted from the patient andsubsequently reintroduced and immobilized in an implant device. Thus,the present invention addresses a need in the art for the real-timecapture and delivery of bioactive molecules and particularly thereal-time utilization of extracted tissues and fluid, whether from anintended recipient or a selected donor organism, as well as materialsthat are synthetically produced or produced from cell cultures.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. However, before the present materials and methods aredescribed, it is to be understood that this invention is not limited tothe particular molecules, compositions, methodologies or protocolsherein described, as these may vary in accordance with routineexperimentation and optimization. It is also to be understood that theterminology used in the description is for the purpose of describing theparticular versions or embodiments only, and is not intended to limitthe scope of the present invention which will be limited only by theappended claims.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. However, in case of conflict,the present specification, including definitions, will control.Accordingly, in the context of the present invention, the followingdefinitions apply:

A. Elements of the Present Invention

As used herein and in the appended claims, the singular forms “a”, “an”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “molecule” is a referenceto one or more molecules and equivalents thereof known to those skilledin the art, and so forth.

In the context of the instant invention, the terms “medical device”,“implant” or “prosthesis” encompasses both devices intended for limitedor temporary introduction (for example, bioerodible tissue scaffolds) aswell as devices intended for long term or permanent insertion (forexample, artificial bone or cartilage). As used herein and in theappended claims, the term “medical device” refers to any apparatus,appliance, instrument, implement, material, machine, contrivance,implant, in vitro reagent, or other similar or related article includinga component party or accessory which is intended for the diagnosis,prevention, monitoring, treatment or alleviation of disease, injury orhandicap. It further encompasses any article intended to affect thestructure or function of the body of humans or other animals, and whichdoes not achieve its principal intended action in or on the bodyexclusively by pharmacological, immunological or metabolic means, butwhich may be assisted in its function by such means. Illustrativeexamples of medical devices contemplated by the present inventioninclude, but are not limited to, bone, cartilage and tooth implants(also prosthetics and substitutes), wound dressings, sutures, staples,anastomosis, vertebral disks, bone pins, suture anchors, haemostaticbarriers, clamps, screws, plates, clips, vascular implants, tissueadhesives and sealants, tissue scaffolds, various types of dressings,intraluminal devices, vascular supports, and other body contactingdevices that may benefit from incorporation with therapeutic materialssuch as therapeutic agents, bioactive molecules, and biological cells ortissues. Also contemplated are devices such as needles, catheters (e.g.,intravenous, urinary, and vascular catheters), stents, shunts (e.g.,hydrocephalus shunts, dialysis grafts), tubes (e.g., myringotomy tubes,tympanostomy tubes), implants (e.g., breast implants, intraocular lens),prosthetics, and artificial organs, as well as cables, leads, wires,electrodes associated therewith (e.g., leads for pace makers andimplantable defibrillators, bipolar and monopolar RF electrodes,vascular guidewires), and devices for isolation and/or concentration ofbioactive materials.

Certain aspects of the present invention permit incorporation ofbioactive materials into the construct of a medical or surgical deviceconstruct without necessitating conjugation, also chemical bonding, withthe device material and as a consequence, any biodegradable and/orbiocompatible material which has value as a part of a medical device,for example a prosthetic implant construct, is of value in the presentinvention.

The present invention makes reference to amphiphilic molecules,particularly amphiphilic molecules that spontaneously assemble into filmmonolayer at the interface between a relatively non-polar material andrelatively polar environment. As used herein, the term film monolayer isinclusive of any plurality of amphiphilic (also amphiphatic orsurfactant) molecules aligned at the interface between a non-polar (alsoless-polar) liquid and polar (also more polar) liquid. Such a monolayercan be present in a wide variety of forms ranging from broken filmswhere alignment is limited to a hydrophobic/hydrophilic alignmentperpendicular to the non-polar to polar interface to more complexcrystalline films and β-pleated sheets. Many of these films are known tothose skilled in the field of surfactants. Monolayers of the presentinvention can also consist of a combination of different amphiphilicmolecules. Such combinations can have value in providing a range ofbinding moieties to a single target cell or molecule, or in providing afilm that can capture a variety of cells or molecules, particularly oneswith synergistic effects. Such combinations can also provide benefit inreducing the cost of a film by achieving a dense molecule layer whereonly some of the molecules have an expensive binding moiety. Byproviding a combination of molecules in a monolayer, the strength of thefilm can also be increased. Typically, the monolayer at the interface isa single molecule thick, however as long as the monolayer at theinterface results in an alignment of binding moieties towards thenon-polar liquid, the monolayer can be more than one molecule thick andstill be of value in the context of the present invention.

Amphiphilic molecules suitable for use in the context of the instantinvention can either be of natural origin or can be synthetic conjugatescreated with specific properties. By definition, an amphiphilic moleculeincludes both hydrophilic and hydrophobic moieties. Whether a givenamphiphilic molecule will form a stable film at the interface between apolar solution, which acts as a solvent, and a non-polar solutiondepends on a variety of factors, including concentration, structure ofthe molecule, temperature and the presence of other amphiphilicmolecules which might tend to increase the film stability. Many growthfactor molecules have been found to be glycoproteins and may either beamphiphilic in nature or be such that they can be conjugated with othermolecules to form an amphiphilic molecule using techniques known tothose skilled in the art. Illustrative methods and materials for formingsuch amphiphilic films are described in WO 2008/154603 (RichardSpedden), the entire contents of which are incorporated by referenceherein.

The present invention makes reference to “bioactive materials” such asstem cells, other biological cells, bioactive molecules, particularlygrowth factors, and other materials of therapeutic value. Bioactivematerials suitable for use in the context of the present invention mayinclude, but need not be limited to, tissues or extracts thereof orother fluids extracted from the patient who is the intended recipient ofthe medical procedure which utilizes the resulting prosthetic device, orbioactive materials from origins other than from the patient. Forcesthat can impart movement of fluid and bioactive materials into anintended biocompatible construct, such as porous prosthetic device caninclude, but not be limited to, pressure or compressive force, gravity,centrifugal force, friction or other mechanical forces, electricalforce, osmotic forces and any other force which one skilled in the artmight employ.

Bone marrow for clinical use is typically obtained as an aspirateextracted from a target patient's bone using a syringe-type device.Often the iliac crest, pelvis, or pelvic bone is used as a source due toits large size and proximity to the surface of the body. In someapplications, the bone marrow is used without modification, but in manycases some form of separation technology, such as centrifugation, isused to concentrate the desired fraction of the bone marrow. Stem cellsand growth factors, are often the target of this separation process.Other bioactive molecules and or other cell types can also be desiredtargets. Cells and molecules of interest are also typically obtainedfrom adipose, also fat, tissue. Any tissue of the body has potential,muscle and nerve tissue and tissues associated with the reproductiveprocess are also of particular interest. Material extracted from thepatient has several advantages over other sources, such as: inherentbiocompatibility, potential for lower cost, providing a broader spectrumof useful compounds which might have synergistic effects and potentiallyreduced regulatory issues or faster regulatory approval. In currentsurgical practice, bone marrow derivatives are typically reintroducedinto the body by injection by syringe into an area of desired activity.Often a porous retention media such as a collagen sponge is used toretain the material in the area.

The term “stem cell” represents a generic group of undifferentiatedcells that possess the capacity for self-renewal while retaining varyingpotentials to form differentiated cells and tissues. Stem cells can betotipotent, pluripotent or multipotent. Derivative stem cells that havelost the ability to differentiate also occur and are termed‘nullipotent’ stem cells. A totipotent stem cell is a cell that has theability to form all the cells and tissues that are found in an intactorganism, including the extra-embryonic tissues (i.e. the placenta).Totipotent cells comprise the very early embryo (8 cells) and have theability to form an intact organism. A pluripotent stem cell is a cellthat has the ability to form all tissues found in an intact organismalthough the pluripotent stem cell cannot form an intact organism. Amultipotent cell has a restricted ability to form differentiated cellsand tissues. Typically adult stem cells are multipotent stem cells andare the precursor stem cells or lineage restricted stem cells that havethe ability to form some cells or tissues and replenish senescing ordamaged cells/tissues. Further information may be found in WO08/007,082, the contents of which are incorporated by reference herein.

The term “progenitor cell” refers to unipotent or multipotent cells,which comprise the stage of cell differentiation between stem cells andfully differentiated cells.

The term “biological cell” refers to any cell capable of performinguseful biological functions in a living organism, particularlyreplication to form a tissue structure. The term as used herein includesstem cells, progenitor cells and fully differentiated cells. Biologicalcells may include cells from the intended host organism or those from adonor organism. Biological cells can include cells from recombinant orgenetic engineering techniques.

The term “bioactive molecules” refers to any molecule which has thecapacity to interact with a living tissue or system in such a way as toexhibit or induce a biological activity in an organism, tissue, organ orcell, either in vivo, in vitro or ex vivo.

Of particular interest in the context of the present invention arebioactive peptides that trigger or regulate biological functions.Illustrative examples of bioactive molecules suitable for use in thecontext of the present invention include, but are not limited to, aregrowth factor proteins, such as TGFβ, BMP-2, FGF and PDGF.

As used herein and in the appended claims, the term “growth factors”refers to the broad class of bioactive polypeptides which controllingand regulating a variety of endogenous biological and cellularprocesses, such as cell-cycle progression, cell differentiation,reproductive function, development, motility, adhesion, neuronal growth,bone morphogenesis, wound healing, immune surveillance and cellapoptosis. Growth factors typically operate by binding to specificreceptor sites on the surface of target cells. Growth factors include,but are not limited to, cytokines, chemokines, polypeptide hormones andthe receptor-binding antagonists thereof. Examples of well known growthfactors include but are not limited to:

-   -   Bone Morphogenic Protein (BMP);    -   Transforming growth factor beta (TGF-β);    -   Interleukin-17;    -   Transforming growth factor alpha (TGF-α);    -   Cartilage oligomeric matrix protein (COMP);    -   Cell Density Signaling Factor (CDS);    -   Connective tissue growth factor (CTGF);    -   Epidermal growth factor (EGF);    -   Erythropoietin (EPO);    -   Fibroblast growth factor (FGF);    -   Glial Derived Neurotrophic Factors (GDNF);    -   Granulocyte-colony stimulating factor (G-CSF);    -   Granulocyte-macrophage colony stimulating factor (GM-CSF);    -   Growth differentiation factor (GDF);    -   Myostatin (GDF-8);    -   Hepatocyte growth factor (HGF];    -   Insulin-like growth factor (IGF);    -   Macrophage inhibitory cytokine-1 (MIC-1);    -   Placenta growth factor (PIGF);    -   Platelet-derived growth factor (PDGF);    -   Thrombocyte concentrate (PRP);    -   Thrombopoietin (TPO);    -   Vascular endothelial growth factor (VEGF);    -   Activin and Inhibin;    -   Coagulogen;    -   Follitropin;    -   Gonadotropin and Lutropin;    -   Mullerian Inhibiting Substance (MIS) also called: Anti-Müllerian        hormone (AMH) Müllerian inhibiting factor (MIF) and Mullerian        inhibiting hormone (MIH);    -   Nodal and Lefty; and    -   Noggin

Molecules which regulate, induce or participate in useful biologicalprocesses in the body, including those listed above, are oftencategorized or classified according to their particular structure orfunction. For example, immunoregulatory proteins secreted by cells ofthe immune system, such as interleukin and interferon, are oftenreferred to as cytokines. Other categories of regulatory moleculesinclude, but are not limited to:

-   -   morphogens (e.g., molecules that regulate or control the        formation and differentiation of tissues and organs);    -   chemokines (e.g., any of a group of cytokines produced by        various cells, as at sites of inflammation, that stimulate        chemotaxis in white blood cells such as neutrophils and T        cells);    -   hormones (e.g., a product of living cells that circulates in        body fluids such as blood and produces a specific, often        stimulatory effect on the activity of cells, usually remote from        its point of origin);    -   receptors (e.g., a molecule present on a cell surface or in the        cell interior that has an affinity for a specific chemical        entity, including both endogenous substances such as hormones        and ligands as well as foreign materials, such as viral        particles, that serves as an intermediary between the        stimulating agent and the downstream physiological or        pharmacological response thereto;    -   receptor-binding agonists (e.g., a chemical substance capable of        combining with a specific receptor on a cell and initiating the        same reaction or activity typically produced by the endogenous        binding substance (such as a hormone); and    -   receptor-binding antagonists (e.g., a chemical substance that        reduces the physiological activity of another chemical substance        (such as a hormone) by combining with and blocking one or more        receptors associated therewith).        However, since the study of the function of the various        regulating moieties in the body is still an emerging science,        the categorization thereof is also evolving. Accordingly, the        present invention is not limited to any one particular class or        category of regulating or stimulating molecules.

As used herein and in the appended claims, the term “growth factors”also refers to precursor forms of growth factors, which are typicallyinactive until they undergo endoproteolytic cleavage, as well assynthesized and recombinant forms which provide part or all of the sameor similar functions as the naturally occurring growth factors.Accordingly, the present invention encompasses precursors, analogues,and functional equivalents of growth factors, provided the resultingmolecules retain some or all of the function of regulating usefulbiological processes in the body, typically by binding to specificreceptor sites on the surface of target cells associated with thewild-type or endogenous moiety.

The term “therapeutic agents” as used herein refers to any molecule,compound or composition having therapeutic potential, more particularlypharmaceutical activity. Examples of particularly useful therapeuticand/or pharmaceutical activities include but are not limited toanti-coagulation activity, anti-adhesive activity, anti-microbialactivity, anti-proliferative activity, and biomimetic activity.

The term “antimicrobial” refers to any molecule which has the capacityto limit or interfere with the biological function of a bacterial,fungal or viral pathogen or a toxin. Antimicrobial is intended to alsoencompass antibacterial, antibiotics, antiseptics, disinfectants andcombinations thereof.

The term “therapeutic materials” refers to any composition whichcomprises any of the following: therapeutic agents, bioactive molecules,stem cells, progenitor cells or biological cells. The term “bioactivesolution” refers to a liquid composition which comprises, in part,bioactive materials.

As used herein, the term “tissue” refers to biological tissues,generally defined as a collection of interconnected cells that perform asimilar function within an organism. Four basic types of tissue arefound in the bodies of all animals, including the human body and lowermulticellular organisms such as insects, including epithelium,connective tissue, muscle tissue, and nervous tissue. These tissues makeup all the organs, structures and other body contents.

As used herein, the term “bone” refers to the rigid organs that formpart of the endoskeleton of vertebrates and function to move, support,and protect the various organs of the body, produce red and white bloodcells and store minerals. One of the types of tissues that make up boneis the mineralized osseous tissue, also called bone tissue, which givesit rigidity and honeycomb-like three-dimensional internal structure.Other types of tissue found in bones include marrow, endosteum, andperiosteum, nerves, blood vessels and cartilage.

Accordingly, the term “tissue” as used herein broadly encompasses allbiological components including, but not limited to, skin, muscle,nerves, blood, bone, cartilage, tendons, ligaments, and organs composedof or containing same.

In the context of the present invention, the term “isolated”, as in, forexample ‘isolated from biological tissues or cells’, refers to anyprocess which separates the therapeutic material of interest from thetissue or cell membranes in a manner which preserves the structure andfunction of therapeutic material of interest. The term “isolated” asused herein is synonymous with the terms “extracted” and “harvested”,for example.

In addition to being isolated, harvested or extracted from naturalsources, therapeutic materials suitable for use in the instant inventioncan also be “derived from” biological sources, for example,synthetically produced or produced by genetically engineered plants andanimals, including bacteria and other microbes, in accordance withwell-known and conventional techniques.

As used herein and in the appended claims, the term “non-polar” refersto a substance or mixture of substances that is relatively unchargedwhen compared to a polar solvent being used. The concept is alsoreflected in the references herein to systems of “differing” or“diverging” polarity. As such, the terms “relatively non-polar”“less-polar” can be interchangeably exchanged herein for the term“non-polar”. The non-polar material is typically water insoluble(hydrophobic). A mixture of non-polar and polar substances can be usedto form the non-polar material of this invention as long as theresulting combination supports the formation of an amphiphilic film whenin the presence of a selected polar solvent. It is further an aspect ofthe present invention that the non-polar liquid can be of a nature whereit can transition to a solid film as taught by Spedden in WO 2008/154603referenced above, the entire contents of which are included herein byreference.

Hereinafter, the present invention is described in more detail byreference to the Examples. However, the following materials, methods andexamples only illustrate aspects of the invention and in no way areintended to limit the scope of the present invention. As such, methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention.

B. Illustrative Embodiments of the Present Invention

It is an object of the present invention to provide materials andmethods which permit a medical device to be exposed to a liquid system,prior to or during surgery, that forms a thin film on the exposedsurfaces, and that such a film contains moieties or molecules havingselective binding affinity for one or more types of biological cells orother bioactive material. In a preferred embodiment, the targetbioactive material is stem cells, due to their broad therapeuticbenefits. However, more differentiated cells and other bioactivemolecules of therapeutic interest may also be targeted. Furthermore, thedevice of interest can be exposed to fluids containing such cells ormaterial either prior to or after introduction of the device in thebody. In a preferred embodiment, a bone marrow aspirate or otherautologous stem cell-bearing fluid is used. However, other fluids,particularly custom designed compositions with commercially availablecells or molecules can also be of value.

To achieve this end, the present invention envisions the use ofamphiphilic molecules wherein the hydrophilic end exhibit specificbinding affinity for a targeted cell or bioactive molecule. Further, inthe context of the present invention, the molecules with the targetingmoieties are, either alone or in combination with other amphiphilicmolecules, allowed to form an amphiphilic monolayer film at theinterface between a non-polar (or relatively less-polar) liquid and apolar liquid, such that the targeting moieties are expressed on thepolar liquid side of the film. Non-polar liquids, typically oils, areknown to readily form thin films on many solid surfaces, such as areencountered in medical implant devices. An amphiphilic film is typicallya single molecule thick (i.e., a monolayer) and tends to stabilize theinterface between a non-polar liquid and a polar liquid. Theseproperties make such a system ideal for medical implant surfaces,particularly the internal surfaces of a porous device, such as aTrabecular Metal™ implant or a woven or other filamentous scaffold.Water, saline solution and most bodily fluids constitute appropriatepolar liquids to allow an amphiphilic film to form and be sustained.

Many non-polar liquids, such as oils, are currently used in medicalapplications. In the preferred embodiment, a fatty acid such as oleicacid is used. Other non-polar materials, such as various oils, siliconeand glycerin, can also be readily used. Biocompatibility is a desirableproperty, since eventually the film may breakdown. Though, the fact thatonly a very thin film of the selected non-polar material is required andthe fact that the amphiphilic film at the interface between polar andnon-polar liquids insulates the selected non-polar material from directcontact with bodily fluids reduces the possibility or degree of anyimmunological reactions to the non-polar liquid.

Amphiphilic molecules of the present invention are typically aconjugation of one or more molecules forming a hydrophobic tail, anoptional spacer molecule and one or more molecules forming a hydrophilichead with desired binding moieties. In a preferred embodiment, thehydrophobic tail is composed of a hydrocarbon chain of the form(CH₂)_(n), where “n” represents the number of hydrocarbon groups and istypically 12 or greater. Lipids and other inorganic and organicmolecules can exhibit appropriate hydrophobic properties. Glycolipids,phospholipids and glycoproteins are in many instances amphiphilic bynature and depending on length and structure, may or may not be suitablefor forming appropriate films. Synthetic polymers, such as polyethyleneglycol (PEG), are often used in the formation of biocompatible,amphiphilic molecules. In US Patent Publication 2007/0141134, Kosakteaches the use of PEG conjugated to aptamers in the formation ofmicelles used as molecular delivery vehicles. The techniques taught inthe formation of these amphiphilic molecules can be used to generatematerials of use in certain aspects of the present invention.

The binding moiety can be any molecular sequence that exhibits thenecessary specificity for the targeted cell or molecule. In thepreferred embodiment, the binding moiety is a nucleic acid targetingmoiety (most preferably an aptamer), where the aptamer has been selectedbecause of a specific binding affinity for a therapeutic cell ormolecule, most preferably a stem cell or other precursor cell.Antibodies and products of phage-display techniques are also of value inproviding the target specific moieties in constructs of the presentinvention. Other molecules with known binding affinities may also beused, though often these do not exhibit as high a degree of specificity.P-selectin, for example, exhibits useful binding moieties for stemcells.

Aptamers are macromolecules composed of nucleic acid that bind tightlyto a specific molecular target that can be economically produced involume. Tuerk and Gold (Science. 249:505-510 (1990)) disclose the SELEX(Systematic Evolution of Ligands by Exponential Enrichment) method forselection of aptamers. In the SELEX method, a large library of nucleicacid molecules {e.g., 10¹⁵ different molecules) can be used forscreening. A desired molecular sequence can be identified through theuse of the technology of systematic evolution of ligands by exponentialenrichment (SELEX). Aptamers are currently used in a range ofbiotechnological and therapeutic applications. They are a competingtechnology to more costly antibodies and have specific advantages overantibodies since they are produced by chemical synthesis (reducing thecost), can be stored and transported easily and have been shown toelicit little or no immunogenicity in therapeutic applications.

Due to the self-aligning nature of an amphiphilic film, binding moietiespresented on the hydrophilic heads of the amphiphilic molecules areexhibited at an extremely high density on the polar liquid side of thefilm. It is an aspect of the present invention that the density of anyspecific binding moiety in the surface can be controlled by theintroduction of other amphiphilic molecules into the polar solutionprior to formation of the film. To form a film with the highest densityof binding moieties possible, only amphiphilic molecules with a specificbinding moiety present are introduced. However, the polar solution canalso be a mixture of amphiphilic molecules with binding moieties andother amphiphilic molecules that do not exhibit binding moieties.Additionally, different amphiphilic molecules can possess differentbinding moieties. The aptamers in a mixture can target a range ofdifferent cell types or molecules. It is an aspect of the presentinvention that a number of different aptamers with moieties targetingdifferent features on the same type of cells can be used in a singlemixture to increase the chance of binding those target cells.

In another embodiment, the present invention takes the form of a kitcomposed of precursor components of the present invention and a methodof using the same. In its simplest form, a non-polar liquid is allowedto coat the surfaces of a medical implant or other device. If thesurface has hydrophobic characteristics, the non-polar liquid willspread to form a very thin film on the surface. Excess non-polar liquidcan be allowed to drain or be flushed from the device. The medicaldevice surfaces are then exposed to a polar solution containing theamphiphilic molecules with nucleic acid binding moieties expressed onthe hydrophilic head of the molecules. Typically, excess amphiphilicmolecule bearing polar solution is drained or flushed from the device soas to reduce the chance of target molecules or cells binding toamphiphilic molecules which are not embedded in surface films. Tomaintain stability of the amphiphilic film, it is preferred that thesurfaces maintain a coating of polar liquid (also, remain wet). In thepreferred embodiment, the device would either be immediately implantedin a patient or exposed to fluids from the patient or a surrogate, orotherwise stored in a polar solution, such as a sterile saline solution.

In another embodiment, present invention contemplates micelle-likestructures surrounding a non-polar liquid core formed utilizingamphiphilic (also bioactive) films such as those described above formedical device surface coatings. As with those films described above,the film can be formed either of molecules exhibiting binding moietiesto cells or therapeutic molecules of interest, or of molecules thatactually incorporate therapeutic molecules in the hydrophilic head ofthe amphiphilic molecule. Further, the non-polar liquid cores in suchstructures can contain additional therapeutic materials.

In another embodiment, the present invention contemplates a solutioncontaining the above micelle, or micelle-like constructs, in which thenon-polar liquid is encased in the bioactive film, and its use inconnection with a porous medical implant device wherein the micellesmigrate into pores of the device. Liquid core micelles often tend tocoalesce over time. These micelles can also be induced to coalesce dueto environmental factors such as increased temperature, mechanicalaction or changes in pH. The coalescing of the micelles in the confinedspace of a pore can increase the interaction between the micelleconstructs and the pore wall, with the result of increased friction thatcan result in the construct being immobilized in the pore. The sameeffect can be achieved if the micelle construct fails and the pore wallbecomes part of the micelle boundary (think of a soap bubble sitting onthe surface of water in the form of half a sphere). In some cases wherethe liquid core micelle fails when in contact with the implant surface,the non-polar liquid will distribute as a thin film on the implantsurface with the amphiphilic film realigning as a barrier between thethin film of non-polar liquid on the implant surface and the surroundingpolar liquid, thus forming a construct described elsewhere.

These effects, which tend to immobilize the films and the non-polarliquid, are of value in the context of the present invention as a meansof introducing and then immobilizing bioactive materials within thepores of the prosthesis. Once implanted in the patient, the biologicalprocesses of the body gradually induce the release the bioactivematerials. Such biological processes include both processes that reactwith and remove bioactive molecules from the films, as well as enzymaticand other processes that might interact with oils or other non-polarmaterials in the constructs.

An important additional feature of the constructs of the presentinvention, particularly those embodiments wherein micelles coalesce inthe pores, is that the film structures not only immobilize theamphiphilic bioactive molecules and the non-polar liquids in the pores,but they also block circulation of polar liquids in the pores,effectively trapping pockets of polar liquids and any materialscontained there-in. This represents an additional aspect of the presentinvention. The polar liquids can contain bioactive molecules as well asbiological cells. Thus water soluble bioactive molecules, which are notnecessarily amphiphilic in nature, can be effectively immobilized in theresulting pockets of polar liquid. Additionally, these pockets cancontain biological cells, and of particular value stem and/or precursorcells.

In addition to micelle-like structures, the films can be contained inoil-in-water or water-in-oil emulsions, both of which are of value inthe context of the present invention. Such emulsions tend to be viscousin nature; they flow under pressure and consequently can be pumped,injected or otherwise introduced into a porous prosthesis, but oncethere, viscous forces will tend to hold the emulsions in place. Thebenefits of these constructs will be the same as those stated for themicelle structures once they are embedded in the pores of theprosthesis.

A suitable non-polar liquid can be chosen on the basis of physicalproperties, such as viscosity, or bio-absorption properties or on anyother basis that achieves the desired results. The non-polar liquid canalso provide therapeutic properties; for example, fatty acids are knownto have some antimicrobial properties, non-polar liquids can be used ascarriers for hydrophobic materials, and oils such as oleic acid arethought to have synergistic effects in combination with growth factorsin stimulating stem cell differentiation and growth. The use of oleicacid and other fatty acids is particularly preferred in the presentinvention.

In another embodiment, the present invention takes the form of agas-liquid foam (also mixture) construct. Such constructs can also beviscous in nature. Due to surface tension properties, the viscouseffects are more pronounced with increased interaction with thesurrounding walls, as in small pores. Consequently, as with emulsions,the foams can be introduced into a porous prosthesis under pressure andthen once in place the viscous forces will tend to immobilize thematerial in the pores until biological processes gradually break downthe foam. As with emulsions, the polar liquid that is entrapped in thepores by the foam can contain bioactive molecules and biological cellsof therapeutic value. In the case of foam used in vivo, the type andquantity of gas is important. In most embodiments of the presentinvention, oxygen will be the preferred gas. Oxygen has the importantcharacteristics of not only being readily absorbed by surroundingtissue, but also being of known value in the promotion of rapidformation of tissue. In excess quantities, oxygen can also serve as anantibiologic. Antibiologic properties can be of value when utilized tocombat infections, which are an issue with any prosthetic implant.Though the antibiologic properties can also hinder tissue development ifthe gas quantity and diffusion rate is not kept to an acceptable level.The appropriate oxygen concentration will differ according toapplication. Tissue oxygen concentrations can range from hypoxic, tonormoxic, to hyperoxic at normal atmospheric pressures and are typicallyreferenced by oxygen tension. A study by Stuart, et al., suggests thatnormalized oxygen tensions that are twice the normoxic level are optimumfor angiogenesis.¹ Any levels above hypoxic are beneficial in modulatingbacterial growth. In the context of the present invention, the oxygen ina foam or otherwise present in a material constrained in the pores of aporous medical implant will, over time, migrate into the surroundingtissue at a rate controlled both by the diffusion characteristics of thefoam or other material and by the partial pressure of oxygen in thesurrounding tissue. The net result is an object of the present inventionwherein, prior to tissue ingrowth, the pores of the implant will havehigh oxygen concentrations, with inherent antimicrobial benefits, and asthe oxygen diffuses out of the pores, the interface at points of tissueingrowth will have an elevated oxygen concentration, promotingaccelerated tissue growth at those points. Zheng et al. (U.S. Pat. No.5,438,041, incorporated by reference herein) describe biocompatibleemulsions with a high oxygen concentration that are applicable to the information of constructs of the present invention. ¹Schugart, Richard C.,Avner Friedman, Rui Zhao, and Chandan K. Sen, Wound angiogenesis as afunction of tissue oxygen tension: A mathematical model, PNAS, Feb. 19,2008, vol. 105, no. 7, 2628-2633.

In yet another embodiment, the present invention contemplates embeddingtwo or more different materials with different viscosities in differentsections of a porous medical implant (also prosthesis) to achieve avariety of effects of value in the context of the present invention. Forexample, a highly viscous material may be embedded in the perimeter zoneof a porous material to act as a hydraulic barrier and constrain a lessviscous material to remain in the center zone of a porous material.Taking a kitchen sponge that has been saturated with water and thencoating it with grease can illustrate this effect, such that the greaseenters the pores on the perimeter of the sponge. The grease will tend toblock the water from draining out of the sponge. The same effect can beachieved using a porous medical implant, any highly viscous,biocompatible material and a less-viscous liquid with bioactivemolecules and/or biological cells. In a preferred embodiment, the two ormore materials with different viscosities are biodegradable orbio-absorbable, though potentially at different rates. In this manner,bioactive molecules or biological cells contained in any or all of thematerials are gradually released into the area of desired therapeuticeffect.

In a particularly preferred embodiment, the highly viscous material onthe perimeter of the medical implant includes antimicrobial materials.This provides an important benefit in combating infection that may havebeen introduced into the patient on the surface of the implant. Theinitial concentration of antimicrobial materials diffusing from thesurface of the devices will gradually be replaced by thebioactive/therapeutic materials contained in the material in theinterior of the device.

When porous medical implants are introduced into a patient, typicallyone or more surfaces function as interfaces with adjoining tissue, suchas bone, with the intent that the tissue will grow into the pores of thedevice along that face or faces, the mating face(s). Accordingly, thepresent invention contemplates that the mating face(s) can have embeddedin the pores a viscous material which either contains bioactivemolecules or serves as a hydraulic barrier to prevent premature flow ofless viscous material in the center of the implant and the othersurfaces of the device can have embedded in the pores a material whichalso serves as a hydraulic barrier to the less viscous material in thecenter of the implant, but which either has a slower absorption ratethan the materials on the mating face(s) or contains the same type ofmaterial but to a deeper depth into the device. The intent of this typeof construct is to permit the therapeutic material in the center of thedevice to have preferential access to the mating face(s) where initialtissue growth is desired.

In a preferred embodiment of the above face specific construct, thesurfaces of the implant device which are not mating face(s) can haveembedded within the pores a solid material with is biodegradable orbio-absorbable. Examples or such materials include, but are not limitedto, PLA, PEG and bio-absorbable waxes. Biodegradable PLA and PEGcompositions, with and without bioactive peptides are known to thoseskilled in the art. An example of a bio-absorbable wax of value in thepresent invention is taught by Nathan, et al., in U.S. Pat. No.7,030,127, “Composition and Medical Devices Utilizing BioabsorbablePolymeric Waxes”, the entire contents of which are included herein byreference.

The embodiments described herein are well suited to the use ofpatient-derived materials, such as bone marrow or adipose tissueextracts. Such extracts can be injected directly into the hydraulicallyisolated center of a porous implant, or, alternatively, the materialscan be compounded with non-polar materials to make micelle or emulsionconstructs that may be more readily immobilized in the pore structuredue to viscous forces.

Accordingly, a porous implant device can be coated or otherwise surfacepore impregnated with a high viscosity, non-water soluble material toform a hydraulic barrier. A portion of one or more surfaces can be leftuncoated so as to permit introduction of a lower viscosity material tofill the interior voids of the device with therapeutic materials.

In another embodiment, the present invention relates to medical implantdevices incorporating the concepts of the present invention, whereinsuch devices are provided in a sterile package, or placed in a separatedevice that permits biological fluids to be passed through the device.Illustrative examples of such devices that may be adapted for use withthe constructs and devices of the instant invention are described inrelated co-pending U.S. patent application Ser. No. 12/498,557, thecontents of which are incorporated by reference herein. Accordingly, theprocess of passing a fluid carrying endogenous material through a porousimplant device, before, during or after the device is implanted in thepatient is considered to be an aspect of the present invention. To thatend, the present invention contemplates the use of external equipment orstructure in which the implant device is held, where there is ahydraulic seal to or surrounding the structure to insure passage offluid through the implant, and with one or more hydraulic connectionsfor introduction of endogenous or other biologic material and one ormore connections hydraulic connections for removal of material.

In another embodiment, the present invention provides materials andmethods enabling therapeutic materials, including, but not limited to,therapeutic agents such as antimicrobials (also antibiotics,antiseptics, disinfectants and combinations thereof), bioactivematerials, including but not limited to growth factors and otherbioactive molecules, and biological materials such as stem cells,progenitor cells and other biological cells to be delivered to a site ofdesired therapeutic use, such as for tissue repair or wound healing.Though not intended as limiting to the application, the materials andmethods of the present invention find particular utility in the contextof surgical procedures, enabling medical personnel to utilizetherapeutic materials that may require immediate use or have restrictivestorage requirements, for example stem cells. The present invention alsoallows therapeutic materials derived from a patient to be used in atherapeutic manner on that same patient, thus reducing the possibilitiesof adverse reactions.

In yet a further embodiment, the present invention provides a kitcomposed of i) a sterile package, or one that can be sterilized, saidsterile package containing ii) one or more porous prosthetic medicaldevices, such devices optionally composed, in part, of surfaces disposedwith surface molecules or non-polar films which represent potentialbinding sites for bioactive molecules, the binding sites based onpotential bonding through chemical conjugation, absorption and/orhydrophobic interaction or other mechanisms for bonding bioactivematerials to substrate materials which are known to those skilled in theart. Furthermore, the kit can provide for introduction into the sterilepackage (e.g., via ports) of target agents comprising bioactivemolecules, cells, other therapeutic or antimicrobial materials orprotective or otherwise useful materials, including, but not limited to,flushing agents, binding agents or coating agents, and that theseprovisions find particular value when configured to permit medicalpersonnel to introduce into the package material extracted from theprospective patient which are thought to include bioactive molecules orcells. Illustrative for introducing target agents include, but need notbe limited to, areas for injection of, or otherwise introduction of,fluids or other materials into the sterile package where the package isthen reseal-able or self-sealing, as might be envisioned by thoseskilled in the art. The kit may optionally further provide for theremoval from the package of excess target agent(s), e.g. via ports, andcan include, but need not be limited to, areas for extraction usinghypodermic needles, one-way valves, deformable polymers which separateand allow flow out when sufficient pressure is exerted on the package,or other directional flow limiting, reseal-able or self-sealing devicesas may be envisioned by one skilled in the art. A properly designedport, can in certain aspects of the present invention serve as both aport for introduction of material as well as a port for removal ofmaterial. In other cases of the present invention, there may beadvantages to a package having a proximal end and a distal end, suchthat the introduction port is located at the proximal end of the packageand the discharge port is located at the distal end of the package. Thisdual port package provides the advantages of a flow through design thatassures that the target agent(s) is well distributed in the package andconsequently has an increased probability of contact with all relevantportions of the device.

In yet a further embodiment, the present invention provides for theconstruction and method of use of a kit configured to permit one or moretarget agents, including, but not limited to, antimicrobial moleculesand/or hydrophobic coating molecules, for example fatty acids, the firstagent(s), to be applied to a portion of the medical device of interestand then permit in a subsequent step the coating or otherwise coveringof the portion with an additional target agent or agent(s), the secondagent(s), including, but not limited to antimicrobial molecules and/orhydrophobic coating molecules, for example fatty acids, or othertherapeutic or protective coatings, in a manner where upon removal ofthe device from the package, the assembled medical device exhibitscertain surface properties of protective or therapeutic use associatedwith the second agent(s) while the interior of the device exhibitsadditional or other properties of therapeutic use related to the firstagents(s). The kit can include any configuration known to those skilledin the art for sequential application of material, including, but notlimited to i) sequential introduction of material in one package zone,and ii) passage of the device through two or more package zones wheredifferent materials can be applied prior to or during removal of thedevice from the package. In the case of a package with two or more zonesfor application of target agents, in certain aspects of the presentinvention, each zone may have agent introduction or removal ports.

The present invention further contemplates methods for applying a targetagent to a portion of a medical device and subsequently coating orcovering the portion with other agents or materials, such a methodcomprising the following steps:

-   -   i) providing a prepared medical device in a sterile package        having a port for allowing sterile passage of at least one        target agent to the device;    -   ii) optionally introducing a binding agent to facilitate binding        of a target agent to the medical device and optionally inducing        excess binding agent to be expelled from the package through a        port;    -   iii) introducing a target agent into the package through a port        to interact with the prepared medical device;    -   iv) inducing a portion of the target agent that fails to bind to        the medical device to be expelled from the package through a        port;    -   v) optionally introducing a flushing material into the package        to assist in diluting and removing excess target agent and        subsequently inducing the flushing material to be expelled from        the package through a port;    -   vi) introducing an additional target agent, agents or coating        molecules into the package through a port or otherwise cause an        additional target agent, agents or coating molecules to be        released in the package in a manner which results in the agents        or molecules coating the medical device.

A package suitable for the application of multiple target agents to amedical device, operable according to the method described above, mayinclude:

-   -   i) a container for receiving the device;    -   ii) a port in communication with the container for allowing        sterile passage of a least one target agent, the first agent(s),        to the prepared device;    -   iii) a second container within the package construct comprising        an additional target agent or agents (including, but not limited        to antimicrobial molecules and hydrophobic coating molecules),        such that the second container can be induced by external        stimulus to release the additional target agents into contact        with the medical device, thus permitting a medical device which        has been exposed to the first agent(s) to be subsequently        exposed to or coated by the second agent(s).

The present invention contemplates the use of bioactive-molecule-bindingamphiphilic moieties, such as those described by Stupp et al. in USPatent Publication 2005/0209145, introducing such moieties into tissuesand their derivatives extracted from a prospective patient (or intendedrecipient), or allografts or xenografts of the same, optionallyconcentrating the tissue solution before or after addition of theamphiphilic moieties, allowing conjugation of bioactive moleculespresent in the tissue with the binding moieties taught by Stupp,allowing the nanowires to form and entangle to form a hydrogel withviscous properties and then introducing the construct into a porousprosthetic device. The resulting construct of nanowire-based hydrogel ina porous prosthetic implant is also an aspect of the present invention,with particular value in constructs incorporating endogenous materialseither bound to or embedded in the hydrogel matrix.

It is further an aspect of the present invention that any or all of theconstructs described as aspects of the present invention can consist ofmaterials mentioned for similar use in the referenced patents, theentire contents of which have been included herein by reference.

C. Methods of Making and Using Embodiments of the Present Invention:

It is an object of the present invention to provide a medical deviceconstruct adapted to incorporate materials extracted from a patient whois the intended recipient of the medical procedure, and that suchextract materials can include, but need not be limited to, bioactivemolecules and stem, progenitor and other biological cells. It is furtherobject of the present invention that the therapeutic molecules and stem,progenitor and other cells be derived from any tissue of the body inwhich the material is present, including, but not limited to, bonemarrow, adipose tissue, muscle tissue and nerve tissue and any fluidsassociated with those tissues. It is further object of the presentinvention that the medical device be adapted to incorporate materialsderived from allografts, xenografts (also zenografts), or syntheticmimics of tissues of the patient who is the intended recipient of themedical procedure, and that the materials can include bioactivemolecules and stem, progenitor and other cells. It is yet another objectof the present invention that molecules and cells of interest suitablefor use in the context of the present invention be derived from productsof the human reproductive system, including autografts, allografts andxenografts of the same.

Due to surface tension and hydrophobic forces, a non-polar (orless-polar) liquid will bind to and distribute across the surface of ahydrophobic solid, and this phenomena will be strengthened by thepresence of a surrounding polar liquid. The forces are such that whenappropriate quantities of non-polar liquid are used, the film can be asthin as a single molecule (e.g., a monolayer). Thus, in the context ofthe present invention, a medical device with hydrophobic surfaces, andfurther provided with a non-polar (or less-polar) liquid film on suchsurfaces, can be exposed to a polar solution in the presence ofappropriate amphiphilic molecules, at which point such amphiphilicmolecules will automatically self-align at the interface between thenon-polar liquid and polar solution. If sufficient amphiphilic moleculesare present, a very closely pack film will develop. “Sufficient” in thiscase is analogous to the Critical Micelle Concentration (CMC) whichdetermines whether micelles will form in a polar liquid with amphiphilicmolecules. The CMC is dependent on many factors, including the nature ofthe amphiphilic molecules, the polar nature of the solvent solution, thetemperature and whether other contaminants or agents are present. Thesame is true for forming self-assembled films on surfaces.

The non-polar (or less-polar) liquids used in the present invention arepreferably biocompatible, more preferably materials which are normallypresent in a prospective patient's body, or analogues, homologues orfunctional equivalents of the materials. Examples of such materials caninclude, but need not be limited to, fats and oils, for example, oleicacid. The use of fatty acids, and particularly oleic acid, as thenon-polar liquid of interest is preferred in the context of presentinvention due to its inherent biocompatibility and the potential synergybetween polyunsaturated fatty acids (PUFA), and specifically oleic acidand bone morphogenetic protein (BMP-2).² ²Ryota Deshimaru, Ken Ishitani,Kazuya Makita, Fumi Horiguchi and Shiro Nozawa, Analysis of fatty acidcomposition in human bone marrow aspirates, The Keio Journal ofMedicine, 54:3-2005, 150-155

Likewise, the amphiphilic molecules used in the present invention arepreferably biocompatible and more preferably include bioactive moleculesderived from a prospective patient. Other amphiphilic molecules whichadd to the stability of the film or provide binding moieties to othercells or bioactive molecules can be introduced. Molecules which providebinding moieties to bioactive molecules can include, among others,heparin and its derivatives and conjugates. Molecules which providebinding moieties to bioactive molecules can include the products ofphage display techniques and antibodies.

The binding of members of the TGF-β cytokine superfamily (growthfactors) to heparin and heparin sulphate containing molecules is known.³In U.S. Pat. No. 6,921,811, the entire contents of which are includedherein by reference, Zomora, et al., teach the coating of medicaldevices with a silyl-heparin complex and a bioactive molecule directlybound to the heparin-activity molecule. In the Zomora patent, thesilyl-heparin complex adheres to the medical device through hydrophobicbonding interaction. Creation of amphiphilic molecules andself-assembled films containing growth factors as taught by Zomora, canfind utility in certain aspects of the novel constructs of the currentinvention that utilize self-assembled films. ³C. C. Rider,Heparin/heparin sulphate binding in the TGF-b cytokine superfamily,Biochem. Soc. Trans. (2006) 34, (458-460) (Printed in Great Britain)

In US Patent Publication 2005/0209145, the entire contents of which areincorporated herein by reference, Stupp, et al., teach the creation ofamphiphilic peptide compounds that incorporate the growth factorrecognition product of a phage display process and the binding of thosecompounds to targeted growth factors. Stupp, et al. also teach the useof these compounds in the creation of self assembled nanofibers ormicelles. Certain of the techniques described by Stupp et al. may findutility in connection with the immobilization bioactive molecules inconstructs of the present invention.

Discher, et al., teach the creation and use of polymersomes and relatedencapsulating membranes in U.S. Pat. Nos. 6,835,394, 7,217,427 and USPatent Publications 2006/0165810 and 2007/0218123, the entire contentsof all of which are incorporated herein by reference. The techniquesdescribed by Discher may find utility in connection with theimmobilization bioactive molecules in constructs of the presentinvention.

Bhaskaran, et al. in U.S. Patent Publication 2008/0058246, the entirecontents of which are incorporated herein by reference, teaches methodsof synthesizing polymer conjugates of growth factor proteins and othercompounds while maintaining a high level of functionality of thesebiological compounds. These techniques may find utility in the contextof the present invention, particularly as a means to immobilizebioactive molecules in constructs of the present invention.

The constructs or methods of conjugating materials can be of value incertain aspects of the present invention, including, but not limited tothe creation of amphiphilic molecules with bioactive components, whichare suited to formation of films at the interface of a polar and aless-polar solution.

Alkan-Onyuksel, et al., teach the creation of micelles and crystallineproducts which incorporate a biologically active amphiphilic (also,amphipathic) compound in U.S. Pat. No. 6,322,810, the entire contents ofwhich are incorporated herein by reference. The methods described in theAlkan-Onyuksel patent may be applicable to certain aspects of thepresent invention.

The use of HBPA-1 heparin gels (heparin and heparin sulphate) to improveangiogenesis is known and is of value in certain aspects of the presentinvention. These heparin gels are thought to recruit and activateendogenous growth factors present at a wound site.⁴ The use of heparinand heparin sulphate in connection with the constructs of the presentinvention is also an aspect of the present invention. ⁴Corral, ClaudioJ. MD; Aamir Siddiqui, MD; Liancun Wu, MD; Catherine L. Farrell, PhD;David Lyons, PhD; Thomas A. Mustoe, MD, Vascular Endothelial GrowthFactor Is More Important Than Basic Fibroblastic Growth Factor DuringIschemic Wound Healing, Arch Surg. 1999; 134:200-205.

In US Patent Publications 2007/0170080 and 2008/0128296, the entirecontents of both of which are incorporated herein by reference, Stopek,et al., teach the construct of a medical device package comprising asealable pouch with a sealed port for introduction of at least one agentto the medical device contained therein. The constructs described byStopek may find utility in connection with certain aspects of thepresent invention. Additionally, the present invention provides forimprovements on the medical device package systems.

In certain aspects of the present invention, the viscosity of anon-polar liquid, or the emulsion or foam thereof, is an importantfactor in limiting the premature migration of a therapeutic from adesired area of retention. Common means of increasing the viscosity ofnon-polar liquids include, but are not limited to, addition of solid,and particularly fibrous, components, gelling of typically oils withcomponents such as pectin, gelatin or aluminum salts of fatty acids suchas aluminum monostearate or distearate, hydrogenation of oils, such asfatty acids, and variation of ratios of components in emulsions andfoams. All of these techniques are of value in the context of thepresent invention. As used herein, the term “foam” refers to any mixtureof gas and liquid wherein both are present in the matrix, and moretypically where the gas is present as discrete zones surrounded byliquids; the presence of a surfactant is not a necessity to form a foamin the present context.

In WO/2005/053767, directed to “CIS-Hydrogenated Fatty Acid Coating ofMedical Devices” the entire contents of which are incorporated byreference herein, De Scheerder, et al. teach the use of hydrogenatedfatty acids as a viscous delivery mechanism for therapeutic agents whenapplied to medical devices, and in particular stents. The materialstaught by Scheerder, et al. find value in certain aspects of the presentinvention. Of particular value in the context of the present inventionis the ability to form materials of differing viscosities.

Stem cells and other precursor cells are known to exhibit a certaindegree of stickiness to other materials. The phenomena of stem cellssticking to the surface of a polystyrene Petri dish is well known tothose practiced in the art. Other molecules are known to bind stem cellsand have been demonstrated as effective means for selectively removingstem cells from biological fluid.⁵ In work done at MIT and theUniversity of Rochester, P-selectin, which exhibits stem cell binding,was immobilized in a polyethylene glycol surface to form a surface whichcan selectively capture stem cells and, particularly in their case,cancer cells.⁶ P-selectin is can be immobilized on biotinylatedsurfaces, such as biotinylated PEG. P-Selectin is an integraltransmembrane glycoprotein expressed in endothelial cells and platelets.As a transmembrane glycoprotein, it also exhibits amphiphiliccharacteristics which make it suited to immobilization at the interfacebetween polar and less-polar surfaces (the technology referencedearlier). ⁵King, Michael, Nichola Charles, Jared Kanofsky, and Jane L.Liesveld, “Using Protein-Functionalized Microchannels for Stem CellSeparation,” Paper No. ICNMM2006-96228, Proceedings of the ASME, June2006.⁶Dougherty, Elizabeth, MIT works toward novel therapeutic device,Tech Talk, Harvard-MIT Division of Health Sciences Volume 52, Number 6,Oct. 24, 2007

P-selectin enhanced surface technology and other surface technologieswhich demonstrate increased binding affinity for stem cells are of valuein the context of the present invention as a coating for the poresurfaces of a prosthetic implant device. Such a construct has particularutility in the present invention in the ability to selectively capturestem cells and other precursor cells from bone marrow and other bodilyfluids which may be passed through the device either prior to or afterintroduction of the device into the patient's body. This process willresult in an increased density of stem and precursor cells populatingthe porous implant with an increased potential for rapid growth oftissue and integration of the implant with adjacent tissues.

Hereinafter, the present invention is described in more detail byreference to the Examples. However, the following materials, methods andexamples only illustrate aspects of the invention and in no way areintended to limit the scope of the present invention. As such, methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention.

EXAMPLES Example 1 Generation of DNA Aptamers Having an Affinity forHuman Mesenchymal Stem Cells (hMSCs)

The surfaces of human mesenchymal stem cells (hMSCs) were targeted forDNA aptamer development. The initial aptamer pool used consisted of 2nmols of random 40-base sequences flanked on either side by known19-base primer sequences (i.e. 5′-forwardprimer-N40-reverseprimer-3′).Reactions and incubations occurred in a selection buffer containing 50mM Tris-HCl (pH ˜7.4), 5 mM KCl, 100 mM NaCl, 1 mM MgCl2, and 0.1% NaN3.These sequences were denatured and renatured by heating to 80 C for 10mins, cooling to 4 C for 10 mins, and then warming to room temperaturefor 20 mins to ensure proper binding structure. Five-fold molar excessesof both yeast tRNA and Bovine Serum Albumin (BSA) were added during thefolding process to lower instances of background binding. The aptamerpool was then incubated in a 1.5 mL low-binding microcentrifuge tubewith a suspension of ˜1 million hMSCs (Passage 2) at 37 C and 5% CO2 for30 mins, before being centrifuged for 10 mins at 1,500 RPMs. Theaspirate, which contained non-binding aptameric sequences, wasdiscarded, and the pellet resuspended in selection buffer with 0.2% BSAand transferred to a new microcentrifuge tube. This was repeated severaltimes, and then the remaining sequences bound to the surfaces of thecells were amplified via polymerase chain reaction (PCR). Forwardprimers were FITC-tagged and reverse primers were biotinylated to assistpurification and analysis. The FITC and the biotin tags were placed onthe 5′ end of the molecule, distanced from the nucleotides via a9-Carbon and an 18-Carbon spacer respectively to prevent stericinterference with the DNA polymerase during PCRs. Post PCR, the mixturewas incubated with streptavidin-coated magnetic beads (Dynabeads® M-280Streptavidin by Invitrogen) to remove biotinylated aptamers from thepool; these were then amplified a second time by PCR and used as theaptamer pool in the next round of SELEX.

To evaluate the changing affinity of the aptamer pool with each round ofSELEX, fluorescence-linked or enzyme-linked methods are employed. MSCsare cultured to confluence in a 96-well imaging plate. Biotinylated andFITC-tagged aptamers from each round are denatured and renatured asdescribed above and incubated in appropriate wells for 30 mins at 37 Cand 5% CO2. In fluorescence-linked assays, wells are aspirated andwashed three times with excesses of phosphate buffered saline (PBS) toremove non-binding sequences. Fluorescent signal per well from theremaining binding sequences is then measured via fluorescence scanner,it being understood that a greater level of signal corresponds to agreater number of binding sequences. In enzyme-linked assays, wells areaspirated (post aptamer incubation) and astreptavidin-horseradish-peroxidase (HRP) conjugate is added to thewells for 30 mins. This is then aspirated and the wells are washed threetimes with excesses of phosphate buffered saline (PBS) to removenon-binding sequences and unbound streptavidin-HRP. An excess ofcolorimetric substrate of the HRP enzyme is then added to each well, andthe plate is covered from light for 20-30 mins to allow the enzyme toreact. Color intensity per well is then measured on a spectrophotometer,it being understood that a greater level of signal corresponds to agreater number of binding sequences.

Example 2 Fluorescently Tagged Aptamers were Immobilized in anAmphiphilic Film of on a Tantalum Metal Medical Implant

Avidin, an amphiphilic molecule, was dissolved in phosphate bufferedsaline (PBS) at a concentration of 200 ug/mL. This solution was thenadded to a liquid olefin (specifically, oleic acid) at a ratio of 1:1 byvolume, and an emulsion was created via vigorous shaking. The surface ofa tantalum medical implant was then exposed to the emulsion such that athin film olefin layer formed on the surfaces of the implant and anavidin nanofilm at the interface between the olefin layer and thesurrounding saline solution. The resulting nanofilm had the activebinding sites of the avidin presented on the PBS side in a denseconfiguration. Additional PBS was used to irrigate the matrix and removeexcess avidin bearing solution.

Aptamers of interest were conjugated with biotin at their 5′ end and afluorescent molecule at their 3′ end. The tantalum metal with theamphiphilic film was then exposed to a solution ofbiotinylated/fluorescent tagged aptamers in PBS such that the biotinconjugated with the immobilized avidin and presented a dense surface offluorescent aptamers at the PBS interface. The matrix was washed againwith PBS to remove unbound sequences and fluorescent scanning was usedto detect the presence of the remaining fluorescently tagged surfaceimmobilized aptamers on the medical implant (FIG. 1B). The experimentwas repeated without the presence of the oleic acid film to demonstratethat the results were the result of an amphiphilic film forming on thenon-polar liquid. No significant fluorescence was detected in thecontrol (FIG. 1A) indicating that aptamer immobilization was due to themechanisms described in the present invention.

Example 3 MSC Capture and Immobilization on a Tantalum Metal MedicalImplant Utilizing Aptamers

Amphiphilic molecules are produced with a hydrophilic head of human MSCspecific aptamers and a hydrophobic hydrocarbon tail of the form (CH2)n,with n in the range of 8 to 24. The molecules are suspended in a sterilenormal saline solution at a density of 240 ug/ml and packaged in a 10 mlsealed glass vial for delivery to surgery, along with a 10 ml sealedglass vial of sterile oleic acid. In surgery, a sterilized TrabecularMetal™ bone implant is place in a dish and the oleic acid is poured ontothe implant in a manner which allows the material to flow by gravitythrough the pores of the device; the implant is further rotated incontact with the oleic acid to provide good distribution and thenremoved from the dish to allow excess material to drain. The implant isthen placed in a close-fitting plastic sleeve and the aptamer solutionis injected into the sleeve. The sleeve is then sealed and agitated fortwo minutes. A sterile saline solution purge is used to remove unboundaptamer in solution. Excess saline solution is drained and immediatelyreplaced by a stem cell rich portion of bone marrow aspirate from thepatient. The implant is then placed into the intended point of repair inthe patient.

Example 4 MSC Capture and Immobilization on a Tantalum Metal MedicalImplant Utilizing Antibodies

Avidin was dissolved in phosphate buffered saline (PBS) at aconcentration of 200 ug/mL and mixed with an equivalent volume of liquidolefin. The mixture was vigorously shaken to create an emulsion, andthis was introduced to the matrices, an 8 mm³ cube of Trabecular Metal™(tantalum) and a glass cover slip. Excess liquid was aspirated and abiotinylated antibody dissolved in PBS was introduced to the matrices ata molar equivalent to the avidin in the film. This antibody was specificto the antigen CD271, or low-affinity nerve growth factor receptor(LNGFR), which is a marker used to characterize human mesenchymal stemcells (hMSCs). Excess liquid was again aspirated, and a suspension of˜2×105 hMSCs (Passage 2) was added to the matrices and incubated with itfor 2 hours. To evaluate the effectiveness of the film, the matriceswere washed four times with an excess of PBS to remove unbound cells.For the glass matrix, remaining cells were then counted via lightmicroscopy. For the tantalum matrix, a luminescence-based assay was usedto calorimetrically quantify remaining cells (CellTiter-Glo® LuminescentCell Viability Assay by Promega). The matrix was incubated with enzymeand colorimetric substrate for 10 mins, before being scanned on aluminometer. The construct of the present invention exhibited a 400%increase in stem cell binding to the substrate over the untreatedtantalum. FIGS. 2 a, b and c are photographic images from this work.

Example 5 Bone Trabecular Metal™ Prosthetic Implant with P-selectinSurface Immobilized in Wax

A bioabsorbable polymeric wax in melted form is passed through atantalum metal prosthetic bone implant which has 80% open area, suchthat the internal surfaces of the implant become coated with melted wax.A solution of P-selectin is then passed through the implant, such thatthe amphiphilic molecules of P-selectin become immobilized in thesurface of the wax. The non-bone mating surfaces of the implant are thento a depth of 4 cm with a highly viscous hydrogenated oleic acid withembedded BMP-2 and FGF. Bone marrow which has been extracted from theprospective patient is then diluted and placed in a device comprisinghydraulic seals to two uncoated faces of the implant and a pump device.The bone marrow is then pumped through the implant such that stem cellsin the marrow are affixed to the P-selectin treated surfaces. Theimplant is then placed in the patient.

Example 6 Bone Prosthetic Implant with Non-bone Mating Surfaces Sealed

The pore surfaces in a tantalum metal prosthetic bone implant which has80% open area, are coated with polyethylene glycol (PEG). The PEGsurfaces are then biotinylated and P-selectin is immobilized on thebiotinylated PEG coated surfaces of the pores of the implant. Thenon-bone mating surfaces of the implant are then sealed to a depth of 4cm with a highly viscous hydrogenated oleic acid with embedded BMP-2 andFGF. Bone marrow which has been extracted from the prospective patientis then diluted and placed in a device comprising hydraulic seals to twouncoated faces of the implant and a pump device. The bone marrow is thenpumped through the implant such that stem cells in the marrow areaffixed to the P-selectin treated surfaces. The implant is then placedin the patient.

Example 7 Micelles in Titanium Dental Implant

Herein, a dental implant composed of an enamel cap over a sinteredtitanium core and root is utilized. BMP-2, FGF and serum albumin areplaced in a sterile saline solution, oleic acid is added to the solutionat a one to three ratio. The resulting mixture is then agitated andsolicited to form micelle structures in the 100 nm to 100 um range. Thedental implant is placed in a device which hydraulically seals theperimeter and provides a liquid entry port on one side of the implantand a liquid discharge port on the other side. The solution iscirculated by pump through the implant for 30 minutes and the implantedin the patient.

Industrial Applicability

Procedures that can be shown to speed recovery and/or increase thesuccess rate of surgical intervention are of high value. Medical devicecoatings that incorporate growth factors and anti-inflammatory moleculesinto surface films to trigger or impair biological responses have beenproposed; however, the effects of such films are limited by thediffusion and subsequent dilution of these molecules over time.Furthermore, while stem cells, growth factors and other bioactivematerials have been shown to provide therapeutic benefits in thetreatment of musculoskeletal conditions, available techniques forretaining and immobilizing such materials in a scaffold of interest aresignificantly limited.

Most useful biological signaling molecules are produced by variousbiological cells; consequently, a far more powerful technology, enablinga more sustained level of signaling molecules in an area beyond thatwhich is achievable by introduction of a set number of molecules duringsurgery, would be to actually immobilize the signaling moleculeproducing biological cells in the target area of interest.Immobilization of stem cells in an area is of particular value sincethey are known to produce both signaling chemicals, which attract otherstem cells to a region, as well as immunomodulatory chemicals, whichreduce swelling and scar tissue formation.

The benefit of a cell-selective, nanofilm-coated implant of the presentinvention is three-fold. First, a cell-selective surface can serve toconcentrate stem (or other targeted) cells from a solution, particularlya solution containing the patient's own cells, eliminating concernsabout incompatibility or infection from allograft tissue. Second, theimmobilization of stem cells will locally enhance the known effects ofthese cells in secreting growth factors to regenerate tissue and reducehealing time. Third, the autologous stem cell-coated nanofilm will serveas a biomimetic scaffold that not only stimulates osteogenesis but alsoreduces immune response to the foreign implant.

Accordingly, the ability to selectively concentrate and immobilizebiological cells on any surface is of great value in evolving fields ofmedicine. The technology of the present invention not only achieves thisend but does so in a manner which is compatible with existing surgicaldevices and techniques and which can be performed by the surgical teamusing autograft stem cells, such as those contained in bone marrow.Additionally, because the present invention is based on the science ofamphiphilic films, the cell binding moieties can be presented at atheoretical maximum density on the surface.

The benefit of a cell-selective, nanofilm-coated implant is three-fold.First, a cell-selective surface can serve to concentrate stem (or othertargeted) cells from a solution, particularly a solution containing thepatient's own cells, eliminating concerns about incompatibility orinfection from allograft tissue. Second, the immobilization of stemcells will locally enhance the known effects of these cells in secretinggrowth factors to regenerate tissue and reduce healing time. Third, theautologous stem cell-coated nanofilm will serve as a biomimetic scaffoldthat not only stimulates tissue growth but also reduces immune responseto the foreign implant.

All patents and publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

While the invention is herein described in detail and with reference tospecific embodiments thereof, it is to be understood that the foregoingdescription is exemplary and explanatory in nature and is intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, one skilled in the art will readily recognize thatvarious changes and modifications can be made therein without departingfrom the spirit and scope of the invention. Other advantages andfeatures will become apparent from the claims filed hereafter, with thescope of such claims to be determined by their reasonable equivalents,as would be understood by those skilled in the art. Thus, the inventionshall be defined not by the above description, but by the followingclaims and their equivalents.

What is claimed:
 1. A method of constructing a target cell-carryingmedical implant device, said method comprising the steps of: a.providing a porous biocompatible medical implant comprising a solidexterior surface having a plurality of pores; b. providing a solutioncomprised of the following components: (i) a non-polar liquid, (ii) apolar liquid and (ii) a plurality of amphiphilic molecules composed ofhydrophobic tail ends and hydrophilic head ends, wherein said head endshave a binding affinity for one or more target cells, further whereinsaid solution components assemble into non-polar-liquid-filled micellesor micelle-like constructs; c. contacting said micelles or micelle-likeconstructs with a solution containing target cells to which thehydrophilic head ends of said amphiphilic molecules have a bindingaffinity and inducing said micelles or micelle-like constructs to bindsaid target cells and form micelle-target cell units; and d. introducingsaid micelle-target cell units into the pores of said implant exteriorsurface so as to yield a medical implant device having target cellsimmobilized within said exterior surface pores.
 2. The method of claim1, wherein at least one of said hydrophilic head ends comprises anucleic acid aptamer.
 3. The method of claim 1, wherein said hydrophilichead ends comprise two or more nucleic acid aptamers having differentstructures and different binding affinities.
 4. The method of claim 1,where said target cell is a stem cell.
 5. The method of claim 1, whereinsaid solution of step (b) further comprises said target cells.
 6. Themethod of claim 1, wherein step (c) is performed in an operating roomenvironment.
 7. The method of claim 1, wherein said target cells areautologous tissue of the patient.
 8. The method of claim 1, furthercomprising the step of inducing a fraction of said micelle-target cellunits to coalesce once introduced in the pores of said medical devicethrough one or more factors selected from the group consisting ofmechanical action, increased temperature, and changes in pH.
 9. Themethod of claim 1, wherein said non-polar-liquid-filled micelles ormicelle-like constructs comprise oil-in-water emulsions or water-in-oilemulsions.
 10. The method of claim 9, wherein said medical device isselected from the list including a wound dressing, a tissue scaffold, avertebral implant, a vascular implant, an intraluminal device, ananastomosis structure, a haemostatic barrier, a surgical suture andbone, cartilage and tooth implants.